Process for producing l-glutamic acid by fermentation of hydrocarbons



United States Patent 3,406,095 PROCESS FOR PRODUCING L-GLUTAMIC ACID BY FERMENTATION 0F HYDROCARBONS Shinichiro Otsuka, 73 Yamashita-cho, Naka-ku, Yokohama-shi, and Ryosuke Ishii, 2 4-chome, Oshimamachi, Kawasaki-shi, both of Kanagawa-ken, Japan; Isamu Shiio, 99 l-chome, Komagome, Toshima-ku, Tokyo, Japan; and Noboru Katsuya, 711 Nakamaruko, Kawasaki-ski, Kanagawa-ken, Japan No Drawing. Filed Mar. 26, 1965, Ser. No. 443,101 Claims priority, application Japan, Mar. 28, 1964,

39/ 17,008 13 Claims. (Cl. 19528) ABSTRACT OF THE DISCLOSURE The yield of L-glutamic acid from the fermentation of a culture medium containing hydrocarbons as principal carbon source bymicro-organisms not requiring biotin for growth is significantly increased by adding substances having penicillin activity in an amount equivalent to 5 to 300 units of penicillin per milliliter of the medium. Various strains of Corynebacterium hydrocarboclastus and Corynebacterium oleophilus are disclosed as effective.

The present invention relates to a method for producing L-glutamic acid by culturing a micro-organism under aerobic conditions on a culture medium containing a hydrocarbon as the main source of assimilable carbon.

It is the primary object of this invention to increase the yield of glutamic acid in such a culture medium and thus to make the commercial use of this process economically feasible.

We have attained this and other objects by adding a penicillin substance to the culture medium at a time after the growth of the micro-organism in the medium has be- 'gun but before it has reached its maximum or stationary phase. The amount of penicillin added to the medium must .be growth-inhibiting but not fatal to the microorganisms in the medium.

A growth-inhibiting amount of penicillin, as used throughout the specification and the claims, is an amount such that the micro-organisms will grow less in the medium after the addition of penicillin than they would grow without such an addition. A fatal dose of penicillin, as used throughout the specification and claims, is an amount such that it destroys or substantially decreases the metabolic activity of the micro-organisms in the medium to produce glutamic acid.

We have found that this addition of penicillin remarkably increases the yield of glutamic acid in fermentative processes using hydrocarbons as a main source of assimilable carbon, independently of the biotin content of the medium or the nutritional requirements of the microorganism employed.

It is known that the addition of penicillin increases the yield of glutamic acid in fermentative processes using biotin-requiring bacteria in a medium containing carbohydrates as a main source of assimilable carbon and an excess of biotin over the optimum amount required. Similar effects counteracting the presence of an excess of biotin in such media have been reported for other antibiotics and for various surface active agents.

We have found, however, that the effect of penicillin is specific in the present method and that such other antibiotics and surfactants are ineffective to increase the yield of glutamic acid in media containing a hydrocarbon as the main source of assimilable carbon.

Except for the addition of penicillin, the culture medium used in the method of the present invention is generally conventional. .Thus, it contains a hydrocarbon or a mixture of hydrocarbons as an assimilable carbon source, an assimilable nitrogen source, inorganic salts and organic growth-promoting substances.

Preferred hydrocarbons include crude petroleum, refined petroleum products and petroleum refining by-products or residual products. The hydrocarbons may be used in this method in the liquid, gaseous, solid or semi-solid state.

Useful liquid petroleum products include kerosene, naphtha, light oil, heavy oil and ligroin.- Useful solid and semi-solid hydrocarbons include the residues from crude oil distillation as well as fractionation residues from hydrocarbon mixtures, such as paraffin, pitch and asphalt. Natural gas and its component compounds may also be employed.

All of these hydrocarbons are insoluble or very sparingly soluble in water. However, they are readily metabolized by micro-organisms in an aqueous culture medium when sufiiciently large surfaces of the carbon source are contacted with the medium. Therefore, if solid hydrocarbons are used, we finely comminute the same and suspend the pulverized solid hydrocarbon in the aqueous medium. If liquid or gaseous hydrocarbons are employed, we vigorously agitate the aqueous medium containing the hydrocarbon to assure intimate contact of the medium with a large surface area of the hydrocarbon. Gaseous hydrocarbons are distributed in the aqueous medium in the form of small bubbles in admixture with air. Emulsifiers and other surfactants may be added, if desired.

The source of assimilable nitrogen, too, it conventional and may include ammonium salts, such as ammonium chloride, sulfate, nitrate or phosphate, soluble nitrates, such as potassium or sodium nitrate, urea, gaseous ammonia or ammonia in aqueous solution, amino acids and protein. The usual range of the nitrogen source concentration is held within about 0.2% to 4% As is also known, higher yields of L-glut-amic acid will be assured by adding to the culture medium growth-promoting amounts of certain minerals, vitamins, amino acids, nucleic acids or related compounds, including yeast extracts, corn steep liquor, peptone, meat extracts, protein hydrolyzates, extracts or pastes from crushed animal or vegetal tissues, and the like.

If desired, supplemental carbon sources may be used, such as well known carbohydrates constituting a source of assimilable carbon, organic acids, polyalcohols, etc.

The addition of penicillin to the culture medium will be effective in increasing the yield of L-glutamic acid with micro-organisms having the following characteristics:

(1) Ability to grow in a medium whose main source of assimilable carbon is a hydrocarbon.

(2) Ability to produce L-glutamic acid by assimilating a hydrocarbon.

(3) A susceptibility to penicillin.

Many micro-organisms are known to have the above three characteristics and may, therefore, be used to advantage in the method of this invention. We have found, however, that the best yields are obtained with bacteria selected from the genus Corynebacterium and the genus Brevibacterium.

Preferred species of Corynebacterium are Corynebacterium hydrocarboclastus and Corynebacterium oleophilus. Vitamin B -requiring strains Corynebacterium hydrocarboclastus M-104 (ATCC No. 15110) and Corynebacterium oleophilus Kp614l4 (ATCC No. 15108) are described in French Patent No. 1,367,815, for instance. They do not require biotin.

As is also conventional, fermentation is carried out at or slightly above room temperature, usually ranging between about 20 C. and 40 C. Aerobic culturing conditions are maintained by shaking, agitating or passing air through the culture medium. This will also enhance the dispersion of the hydrocarbon suspended in the medium.

The pH of the medium is held between 4 and 9. Since the hydrogen ion concentration will increase in the fermentation broth as L-glutamic acid and other organic acids are produced therein, and as ammonia is consumed, if ammonium salts are used as the nitrogen source, a phosphate salt may be added to maintain an optimum pH value- Other pH controlling agents are such alkaline materials as sodium hydroxide, gaseous or liquid ammonia, or urea, the latter serving also as additional nitrogen sources.

The fermentation process usually is completed within one to five days.

Penicillin may be added to the culture medium in its pure form as well as in the form of its salts or other derivatives. Thus, the term penicillin, as used herein, includes all the members of the penicillin group, such as benzyl penicillin, phenoxymethyl penicillin, phenoxyethyl penicillin, phenoxypropyl penicillin, dimethoxyphenyl penicillin, aminobenzyl penicillin, methylphenylisoxazolyl penicillin, the sodium and potassium salts as well as products of decomposition thereof, all of which have a penicillin-like activity against the micro-organism used in the fermentation process. All of these useful penicillin substances have in common the property of inhibiting the growth of the micro-organism, i.e. the micro-organism is susceptible thereto.

Since the fermentation is carried out with an aqueous culture medium, we prefer to use a water-soluble penicillin salt. Aqueous solutions of potassium and sodium penicillin are most convenient.

The penicillin must be added to the culture medium after the initial growth of the micro-organism has begun but before this growth reaches its maximum, at which point it levels off to remain in a stationary growth phase. Best results are obtained if the penicillin is added during the logarithmic growth phase of the micro-organism.

The growth-inhibiting amount of penicillin added to the culture medium will depend on such factors as the type of micro-organism in the medium, the composition of the medium, the incubation conditions, the type of penicillin, and the time at which the penicillin is added. However, the amount must be suflicient to inhibit the growth of the micro-organism noticeably and must be insufficient to destroy or substantially decrease the metabolic activity of the micro-organism necessary to produce glutamic acid. Less than the indicated minimum amount of penicillin, i.e. an amount permitting the micro-organism to grow in the medium as though no penicillin had been added, will produce no substantial effect on the glutamic acid yield. On the other hand, an excess amount of penicillin beyond the indicated one will subject the micro-organism to a fatal dose, i.e. it will make it useless for producing glutamic acid.

While those skilled in the art will adjust the exact amount of penicillin readily on the basis of this teaching and without undue experimentation, depending on the indicated factors, we have found an addition of about 5 to 300 units of penicillin per milliliter of culture medium to be elfective.

The addition of penicillin according to this invention considerably increases the glutamic acid yield and this increase may be as much as tenfold, or more, over that obtained in the identical process without penicillin addition.

L-glutamic acid may be recovered from the fermentation broth by conventional methods, such as by means of ion exchange resins or crystallization, after residual amounts of hydrocarbon have been removed, for instance by centrifuging.

The method of the invention is further illustrated by the following examples but it will be understood that the invention is not limited thereto.

Example 1 A medium of the following composition was prepared:

Na HPO grams 6.8 KH2PO4 d MgSO do 0.5 NaCl do 0.5 NH Cl do 5.0 FeSO mg 10 MnSO mg 8.2 Vitamin B HCl mg 1 These substances were dissolved in distilled water to make up 900 ml. of an aqueous medium. After adjustment of the pH value of the medium to 7, portions of 22.5 ml. of the medium were introduced into 500 m1. flasks and 2.5 ml. of kerosene were introduced into each flask. The medium in each flask was then sterilized at C. for 10 minutes by autoclave.

The microorganisms shown in Table 1 were cultured on a bouillon agar slant at 30 C.,for 48 hours. Then the above-mentioned medium in each flask was inoculated with the thus obtained seed culture and the culture was carfied out at 30 C., with shaking.

TABLE 1 L-glutamic acid accumulated (ma/1.)

Microorganism employed As the pH value of the medium fell during the fermentation, it was controlled between 7.0 and 7.2 by adding 2 N aqueous ammonia twice a day.

24 hours after inoculation, an aqueous solution of potassium benzyl penicillin was added to one group of flasks to give a concentration of 30 units per ml. of culture medium while a test group of flasks was left without penicillin addition. The culture was then continued under the same conditions as mentioned above in all flasks.

After 72 hours, the culture broth was assayed and the L-glutamic acid content shown in Table 1 was found.

Example 2 The fermentation was carried out, employing the same conditions and medium as described in Example 1, but employing different concentrations of penicillin and different addition periods of it, as shown in the following Table 2.

The microorganism employed was Corynebacterium hydrooarboclastus M-104 (ATCC N0. 15,110). The L- glutamic acid concentration accumulated in the culture broth after 72 hours of fermentation is shown in the Table 2.

TABLE 2 Addition period, Concentration of L-glutamic acid concentration of accumulated (man/l.) penicillin added (units/ml.) After 24 hours After 48 hours from inoculation from inoculation Example 3 The fermentation was carried out under the same conditions as described in Example 1 by employing Corynebacterium hydrocarboclastus M104 (ATCC No. 15,110), but adding 30 units/ml. of penicillin to the medium after 16 hours from inoculation and employing nirtogen sources shown in the following Table 3.

The concentrations of L-glutamic acid accumulated in a broth after 72 hours incubation are shown in Table 3. In this example, the concentration of nitrogen source was set so that the concentration of nitrogen is equal to that of 0.5% of ammonium chloride, and the pH value of the medium was kept in a range of from 4 to 9 by addition of 2 N solution of sodium hydroxide.

TABLE 3 Nitrogen source Concentration of L-glutamic employed: acid accumulated (mg/l.) Sodium nitrate 860 Ammonium nitrate 1,420 Urea 180 Ammonium chloride 1,480

Example 4 TABLE 4 Concentration of Irglutamic acid accumulated (mg/1.) Hydrocarbon employed No penicillin Penicillin was added was added n-Undecane- 300 1, 500 n-Dodecane 100 1, 900 n-Tetradecan 300 4, 000 n-Hexadecane 200 2, 800

No'rE.-The concentration of the hydrocarbons employed was volume percent.

While the present invention has been described in connection with certain specific examples, it will be understood that many variations and modifications may occur to the skilled in the art, particularly after benefitting from this teaching, without departing from the spirit and scope of the invention as defined by the appended claims.

We claim:

1. In a method of producing L-glutamic acid by culturing a microorganism on a culture medium under aerobic conditions, the medium containing a hydrocarbon as a main source of assimilable carbon, a source of assimilable nitrogen and microorganism growth-promoting amounts of inorganic salts, and the microorganism having the ability of growing in said medium in the absence of biotin and of assimilating the hydrocarbon to produce L-glutamic acid, and being susceptible to penicillin, the improvement of adding a growth-inhibiting amount of a substance selected from the group consisting of penicillin and penicillin compounds to said culture medium after the initial growth of the microorganism has begun in the medium but before said growth reaches its maximum, and recovering L-glutamic acid from the cultured medium.

2. In the method of claim 1, the microorganism being selected from the genus Corynebacterium.

3. In the method of claim 2, the microorganism being selected from the species consisting of Corynebacterium hydrocarboclastus and Corynebacterium oleophilus.

4. In the method of claim 3, the microorganism being selected from the strain Corynebacterium hydrocarbocla'stus M-104 (ATCC No. 15110).

5. In the method of claim 1, the hydrocarbon being selected from the group consists of crude oil kerosine, naphtha, light oil, heavy oil and ligroin.

6. In the method of claim 1, the penicillin compound being selected from the group consisting of benzyl penicillin, phenoxymethyl penicillin, phenoxypropyl penicillin, phenoxyethylyl penicillin, aminobenzyl penicillin, methylphenylisoxyazolyl penicillin, their sodium and potassium salts and their decomposition products.

7. In the method of claim 1, the penicillin substance being added to the medium in the range of 5 to 300 units of penicillin per milliiter of the medium.

8. In the method of claim 1, the penicillin substance being added during the logarithmic growth phase of the microorganism.

9. In the method of claim 1, the culturing of the microorganism being carried out at a temperature range of about 20 C. to 40 C. and in a pH range of 4 to 9.

10. In the method of claim 3, the microorganism being selected from the strains consisting of Corynebacterium hydrocarboclastus M104 (ATCC No. 15,110), Corynebacterium hydrocarboclastus A-137 (ATCC No. 15,- 968), Corynebacterium hydrocarboclastus p-9-1474 (ATCC No. 15,960), Corynebacterium hydrocarboclastus p81l484 (ATCC No. 15,961), Corynebacterium hydrocarboclastus p821474 (ATCC 15,962), Corynebacterium olephilus KP6-1414 (ATCC No. 15,108) Corynebacterium oleophilus KP-10-1399 (ATCC NO. 15,963).

11. In a method of producing L-glutamic acid, the step of culturing a micro-organism on a culture medium under aerobic conditions, the medium containing a hydrocarbon as a main source of assimilable carbons, a source of assimilable nitrogen, growth promoting amounts of inorganic salts, and a growth inhibiting agent having penicillin activity of 5 to 30 penicillin units per milliliter of said medium, the medium being substantially free from biotin, the microorganism being selected from the strains consisting of Corynebacterium hydrocarboclastus M-104 (ATCC No. 15,110), Corynebacterium hydrocarboclastus A-137 (ATCC No. 15,968), Corynebacterium hydrocarboclastus p91474 (ATCC No. 15,960), Corynebacterium hydrocarboclastus p8-11484 (ATCC No. 15,961), Corynebacterium hydrocarboclastus p82-1494 (ATCC 15,- 1962), Corynebacterium olephilus KP-61414 (ATCC No. 15,108), Corynebacterium oleophilus KP-l0-1399 (ATCC No. 15,963).

12. In the method of claim 11, the growth inhibiting agent being added to said medium after growth of said microorganism in said medium has begun, but before said growth reaches its maximum.

13. In the method of claim 12, said hydrocarbon being an alkane having a straight carbon chain of eleven to sixteen carbon atoms.

References Cited UNITED STATES PATENTS 3,080,297 3/1963 Phillips et al. 47 3,222,258 7/1965 Iizuka et al 195-29 OTHER REFERENCES Shiio et al., Journal of General Applied Microbiology, vol. 9, No. 1, pp. 23-30, 1963.

LIONEL M. SHAPIRO, Primary Examiner. 

